During the late stages of the virus life cycle, HIV-1 packages two copies of the viral genome into each assembling HIV-1 virus particle. Although this process is critical for production of infectious virus, where and how packaging occurs in cells remains unclear. Because so little is known, it is generally assumed that packaging is initiated when Gag and HIV-1 genomic RNA (gRNA) randomly find each other anywhere in the cytoplasm. In contrast, our findings argue for a very different model, in which gRNA packaging is initiated within a specific subcellular complex through a highly regulated process. Our preliminary data reveal that prior to and during assembly, non-translating gRNA is largely found in cellular complexes termed RNA granules. Moreover, we find that a subclass of RNA granules containing gRNA and Gag can be isolated using an antibody to the cellular ATPase ABCE1. Thus, ABCE1 is a marker for the RNA granules in which packaging takes place. Additionally, we have identified three categories of ABCE1-containing packaging granules: those involved in early, intermediate, and late stages of packaging. Here we will use our ability to isolate RNA granules representing different stages in HIV-1 packaging to define molecular events involved in initiation and completion of gRNA encapsidation. In Aim 1, inducing Jurkat cells to synchronously express latent HIV-1 genomes will allow us to follow the kinetics of gRNA trafficking through each of the packaging granules we have identified and into released virus. We will also confirm the physiological relevance of HIV-1 packaging granules using HIV-1 infected primary human T cells. In Aim 2, we will identify components of HIV-1 packaging granules. This will be done using a hypothesis-driven approach, in which we will test for viral components expected to be present during packaging (e.g. lysyl tRNA primer and a dimeric form of gRNA), and using a discovery- based approach to define the cellular proteome and RNAome of early, intermediate, and late packaging granules. Finally, in Aim 3, we will use innovative crosslinking-immunoprecipitation (CLIP) technology to understand conformational changes that occur during packaging. Others recently examined all Gag in the cytoplasm vs. at membranes using CLIP and found that the number of Gag-gRNA contact sites increase dramatically during packaging. Here, we propose to use CLIP to determine whether HIV-1 packaging granules are the sites in which Gag proteins make those changing gRNA contacts. We will also use CLIP to identify host proteins that make contact with gRNA and are therefore likely to promote encapsidation. Studies in Aim 3 will also identify host proteins that contact Gag, thereby helping to define conformational changes that Gag undergoes during packaging. Together, the proposed aims will greatly advance our understanding of the molecular basis of packaging in cells, and will provide insight into recently discovered small molecules that inhibit virus-specific RNA granules.